Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein secreted by macrophages, fibroblasts, and endothelial cells originally identified by its ability to stimulate the survival, proliferation, and differentiation in vitro of predominantly neutrophilic granulocytes from bone marrow progenitors (Nicola, N. A., Annu. Rev. Biochem. (1989) 58:45). The capacity of G-CSF to regulate in vivo granulopoiesis is supported by animal and clinical studies, which demonstrated a reversible rise in circulating neutrophil levels in response to administered recombinant G-CSF (Gabrilove, J. L. et al., N. Engl. J. Med. (1988) 318:1414). G-CSF has pleiotropic effects on mature neutrophils, enhancing their survival and stimulating functional activation, including induction of neutrophil alkaline phosphatase (Sato. N. et al., J. Cell. Physiol. (1988) 37:272) and high affinity IgA F.sub.c receptors (Weisbart, R. H., et al., Nature (Lond.) (1988) 332:647), priming for respiratory burst (Nathan, C. F. Blood (1989) 73:301) and increased chemotaxis (Wang, J. M., Blood (1988) 72:1456). G-CSF effects have also been observed on hematopoietic cells that are not committed to the granulocyte lineage, for example, stimulation of the proliferation on monocytic differentiation in vitro of some myeloid leukemic cells (Geissler, K., J. Immunol. (1989) 143:140) and the proliferation in vitro of some multipotential hematopoietic precursors (Ferrero, D., Blood (1989) 73:402).
G-CSF activates intracellular processes by binding to the granulocycte colony stimulating factor receptor (G-CSF receptor) (Demetri, G. D. and Griffin J. D., Blood 1991, 78, 2791; Avalos, B. R. Blood, 1996, 88, 761). The G-CSF receptor is single transmembrane cytokine cell surface receptor composed of three domains: an extracellular ligand binding domain, a transmembrane domain, and an intracellular signal transduction domain. It is now clear that signal transduction by cytokines is accomplished by ligand-mediated receptor dimerization (Ullrich, A. and Schlessinger, J., "Signal transduction by receptors with tyrosine kinase activity", Cell, 61" 203-212 (1990); Kishimoto, T., Taga, T., and Akira, S., "Cytokine signal transduction", Cell, 76: 253-262 (1994); Heldin, C. H., "Dimerization of cell surface receptors in signal transduction", Cell, 80: 213-223 (1995); Lemmon, M. A. and Schlessinger, J., "Regulation of signal transduction and signal diversity by receptor oligomerization", Trends Biol. Sci., 19: 459-463 (1994)). G-CSF binds to two receptor subunits resulting in homodimerization, an event that promotes activation of cytoplasmic tyrosine kinases that associate with the intracellular domain of the recptors. This tyrosine kinase activity then initiates a cascade of intracellular processes.
Administration of recombinant G-CSF to patients suffering from neutropenia due to various causes indicated that G-CSF is beneficial as an adjuvant in chemotherapy and in bone marrow transplantation (Morstyn, G., et al., Trends Pharmacol. Sci. 10, (1989) 154-159). G-CSF activity is also associated with mobilization of hematopoietic stem cells from the marrow to the peripheral blood. (Haylock et al., Blood 89:2233-2258, 1997).
Despite the success of recombinant G-CSF in producing an agonist response at the G-CSF receptor, it is not considered an ideal pharmaceutical treatment. Lack of oral bioavailability and a limited serum half-life limit the desirability and efficacy of recombinant G-CSF as a pharmaceutical agent. Consequently, the need exists to provide improved ligands which have agonist properties towards the G-CSF receptor.
The human G-CSF receptor was first sequenced in 1990 (Fukunaga, R., Seto, Y., Mizushima, S., and Nagata, S. Three different mRNAs encoding human granulocyte colony-stimulating factor receptor Proc. Natl. Acad. Sci. U.S.A. 87(22):8702-8706, 1990) and had already been characterized by radioligand studies on purified human blood neutrophils by 1986 (Nicola N A; Vadas M A; Lopez A F, J-Cell-Physiol. 1986, 128 501-9). Notwithstanding the fact that assay tools have been available for over a decade, only one application (PCT/US97/08864) describes small organic molecules which exhibit G-CSF mimetic activity. This application does not mention zinc chelated small organic molecules.
As disclosed herein it has unexpectedly been discovered that certain selected zinc chelated receptor ligands have agonist properties towards the G-CSF receptor.
This discovery, that zinc chelated small organic molecules exhibit agonist activity towards the G-CSF receptor is particularly surprising in view of the finding that the natural ligand for the G-CSF receptor (i.e. G-CSF or recombinant G-CSF) does not utilize zinc chelation during activation. This finding is exemplified and further discussed in the examples below.